Acute and chronic effects of environmental realistic concentrations of simvastatin in danio rerio: evidences of oxidative alterations and endocrine disruptive activity

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Highlights

  • Simvastatin exposure in zebrafish affected behaviour through hiperactivity.

  • Simvastatin’s antioxidant properties caused a decrease in biochemical biomarkers.

  • This antihyperlipidemic drug did not act as a reproductive disruptor.

Abstract

Due to their wide use, pharmaceuticals can be discarded, metabolized and excreted into the environment, potentially affecting aquatic organisms. Lipid-regulating drugs are among the most prescribed medications around the world, to control human cholesterol levels, in more than 20 million patients. Despite this massive use of lipid-regulating drugs, particularly simvastatin, the role of these drugs is not fully characterized and understood in terms of its potential toxicological effects at the environmental level. This work intended to characterize the toxicity of an acute (120 h post-fertilization) and chronic (60 days) exposure to the antihyperlipidemic drug simvastatin (in concentrations of 92.45, 184.9, 369.8, 739.6 and 1479.2 ng L−1), in the freshwater species zebrafish (Danio rerio). The concentrations hereby mentioned were implemented in both exposures, and were based on levels found in wastewater treatment plant influents (11.7 ± 3.2 μg L−1), effluents (2.65 ± 0.8 μg L−1) and Apies River (1.585 ± 0.3 μg L−1), located in Pretoria, South Africa and, particularly in the maximum levels found in effluents from wastewater treatment plants in Portugal (369.8 ng L−1). The acute effects were analysed focusing on behavioural endpoints (erratic and purposeful swimming), total distance travelled and swimming time), biomarkers of oxidative stress (the activities of the enzymes superoxide dismutase, catalase, glutathione peroxidase), biotransformation (the activity of glutathione S-transferases) and lipid peroxidation (levels of thiobarbituric acid reactive substances). Animals chronically exposed were also histologically analysed for sex determination and gonadal developmental stages identification. In terms of acute exposure, significant alterations were reported in terms of behavioural alterations (hyperactivity), followed by a general reduction in all tested biomarkers. Also, the analysis of chronically exposed fish evidenced no alterations in sex ratio and maturation stages. In addition, the analysis of chronically exposed fish evidenced no alterations in terms of sexual characteristics, suggesting that the chronic exposure of Danio rerio to simvastatin does not alter the sex ratio and maturation stages of individuals. This assumption suggests that simvastatin did not act as an endocrine disruptor. Moreover, the metabolism, neuronal interactions and the antioxidant properties of SIM seem to have modulated the hereby-mentioned results of toxicity. Results from this assay allow inferring that simvastatin can have an ecologically relevant impact in living organisms.

Introduction

The use of pharmaceuticals to prevent and treat several diseases has led to a worldwide increase in their consumption and environmental presence (Jelic et al., 2011; Kaczala and Blum, 2015; Ebele et al., 2017). Some pharmaceuticals are discarded directly into the wild or sent to wastewater treatment plants (WTTPs) from which are eliminated after treatment, being ultimately discarded into receiving waters (Boxall, 2004). The results of previous investigations have shown that classic WWTPs cannot completely remove many pharmaceuticals from residual waters (Paíga et al., 2019). Physicochemical properties of drugs, such as biodegradability, lipophilicity, solubility, photosensitivity, and volatility, as well as operation and climate conditions during the process of treatment, can affect the removal rate of pharmaceuticals (Boxall, 2004; Gracia-Lor et al., 2012). Thus, their occurrence has been detected worldwide, in sewage treatment plants, seawater, surface water and groundwater (Nikolaou et al., 2007).

When in the environment, these substances may potentially affect aquatic organisms and cause short and long-term effects, including toxicological interactions with varied pathways and receptors (El-Saad and Elgerbed, 2010). By attaining this purpose, drugs may damage the proper functioning of the liver and the central nervous system (CNS), affecting behaviour (El-Saad and Elgerbed, 2010; Monat-Descamps and Deschamps, 2012; Omar and Mahmoud, 2017). One type of behavioural parameter that may be measured is the swimming patterns of fish, as the individuals can develop altered locomotory responses, and the frequency of swimming movements and duration of activity can change due to the exposure to xenobiotics (Sharma, 2019). Movement analysis is often conducted by automated biomonitoring systems because of their sensitivity, involving the quantification of swimming patterns and videography (Dagneaux et al., 2009).

The components and extent of biological response to contaminants can also be measured by biochemical biomarker assessment (Jemec et al., 2010). One of the most recurrent responses to chemical compounds involves their metabolism through the activation of phase I of enzymatic complexes, namely cytochrome monooxygenases P450 enzymes (CYP450) (Holth et al., 2008; Sharma et al., 2012). Commonly, the poor coupling of the CYP450 catalytic cycle results in the continuous production of reactive oxygen species (ROS), that are relevant in signalling pathways but may be responsible for oxidative stress, where the production of ROS surpasses the efficacy of antioxidant defences (Betteridge, 2000; Ray et al., 2012; Banerjee et al., 2016). Some examples of biomarkers of oxidative stress are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). SOD constitutes protection against the radical superoxide (O2) toxic effects, as it catalyzes its dismutation into hydrogen peroxide (H2O2) and molecular oxygen (O2). As H2O2 is continuously produced in the organism, two enzymes ensure its removal, namely CAT and GPx (Lumb, 2017). CAT is part of the antioxidant defence system that occurs in peroxisomes (Modesto and Martinez, 2010), subcellular structures where many enzymes that are responsible for the production of H2O2 are located (Djordjević, 2004). GPx also acts against H2O2, by the conversion of reduced glutathione (GSH) and H2O2 into water and oxidized glutathione (GSSG) (Blondet et al., 2018).

One example of phase II enzymes is the group of metabolic isoenzymes glutathione S-transferases (GSTs), which act by conjugating GSH with electrophilic centers, resulting in significant water solubility of the newly formed complex, and the ultimate detoxification of xenobiotics. In that way, GSTs prevent the interaction of xenobiotic substrates with nucleic acids and cellular proteins (Dzoyem and Eloff, 2014). GSTs can also transport proteins and some of their isoenzymes can reduce organic hydroperoxides and are responsible for the isomerization of unsaturated substances, protecting the organism against oxidative stress, and therefore, oxidative damage (Rahman, 2007; Smith et al., 2013; Dzoyem and Eloff, 2014).

Lipid peroxidation can result because of the depletion or inefficacy of the previous oxidative damage preventive measures (Sharma et al., 2012). Free radicals interact with lipids present in membranes, particularly unsaturated fatty acids (Halliwell, 2009). As secondary products, various aldehydes are formed, such as malondialdehyde (MDA)-like substances, whose levels are used as one of the most comprehensive indicators of the total amount of final products of lipid peroxidation (Lykkesfeldt, 2007; Ayala et al., 2014). The quantification of such substances may be performed by measuring the levels of TBARS (Dzoyem and Eloff, 2014).

Some xenobiotics, known as Endocrine-Disrupting Chemicals (EDCs), act by favouring endocrine disruption (Voss et al., 2005; Bogers, 2008). Some examples of this type of EDCs of anthropogenic origin are pharmaceuticals (Metzler, 2006). One of the most sensitive and important effects caused by exposure to EDCs are deleterious histological modifications of gonadal tissues, which may result in changes in sex ratio and maturation stages of affected individuals (Bogers, 2008). Danio rerio was suggested as a model organism for the assessment of EDCs effects in terms of gonad development (Örn, 2006).

Lipid-regulating drugs are one of the most prescribed medications around the world, to control human cholesterol levels (Davidson, 2002). There are several lipid-regulating drugs, such as fibrates, inhibitors of absorption of cholesterol, nicotinic acid, and fish oil derivatives, but statins are the predominant group (Rang et al., 2007). Statins exert their therapeutic activity by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (Davidson, 2002; Rang et al., 2007). HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonic acid, a cholesterol precursor (Chavan-Gautam et al., 2018). Thus, the most evident effect of statins is the reduction of plasma (low-density lipoproteins) LDL, some reduction in plasma triglycerides, and an increase in HDL (high-density lipoproteins) (Rang et al., 2007). One of the most sold statin worldwide is simvastatin (SIM). SIM can also increase mitochondrial and peroxisomal β-oxidation of fatty acids (Park et al., 2016). The degradation of fatty acids has the primary goal of the formation of acetyl-CoA, to serve as a carbon and energy source, essential for the metabolism of the individual. Thus, it is possible to expect an increase in locomotion of individuals exposed to this substance (Bhagavan and Ha, 2015). The antioxidant properties of SIM should also decrease erratic behaviour, through the reduction of stress and anxiety-like behaviour (Bouayed, 2011; Hassan et al., 2014; de Carvalho et al., 2019). β-oxidation and the Krebs cycle are closely related to the electron transport chain. In turn, the final electron acceptor of the electron transport chain consists in O2, which leads to the production of H2O, and ultimately to the formation of ROS, such as O2, HO and H2O2 (Speijer et al., 2014). The production of ROS can, therefore, lead to oxidative stress, and ultimately to oxidative damage, thus altering the activity of such enzymes. However, the antioxidant properties of SIM could counteract this effect, by decreasing the activity of these enzymes (Hassan et al., 2014). HMG-CoA reductase inhibitors, such as statins, are thought to affect sex hormone biosynthesis, inhibiting the synthesis of cholesterol, a precursor of estradiol and androstenedione, which should affect sex ratio and maturation stages of individuals exposed to SIM (Ser et al., 2010).

Considering its wide and continuous use by humans, the occurrence of SIM in wastewater all over the world has been reported in various studies, in the range of 0.04−718 ng L−1, in multiple countries, such as Greece, with concentrations reaching 718 ng L−1, in wastewater influents (Papageorgiou et al., 2016); in the UK, reaching 115 and 5 ng L−1, in wastewater influents and effluents, respectively (Kasprzyk-Hordern et al., 2009; Boleda et al., 2011; Martín et al., 2011; Papageorgiou et al., 2016; Tete et al., 2019); and in Spain, 7.5 ng L−1, in a drinking water treatment plant (Boleda et al., 2011). Samples from the river Danube also reported a concentration of 0.04 to 0.7 ng L−1 (Martín et al., 2011). In Pretoria, South Africa, the concentrations of simvastatin found for WWTP influents, effluents and in Apies River, were 11.7 ± 3.2 μg L−1, 2.65 ± 0.8 μg L−1 and 1.585 ± 0.3 μg L−1, respectively (Tete et al., 2019). Particularly at the national level, the maximum level found in effluents from WWTPs in Portugal was 369.8 ng L−1 (Salgado et al., 2010). The continuous widespread of this pharmaceutical, which may have toxicological effects in organisms, constitutes a major concern, that was assessed in this study.

The main goal of this work was to evaluate the toxic effects in Danio rerio resulting from an acute [120 h post-fertilization (hpf)] and chronic [60 days post-fertilization (dpf)] exposure to SIM in ecologically relevant concentrations through behavioural analysis (erratic and purposeful swimming, total distance and swimming time), biochemical markers of oxidative stress (determination of the activity of the enzymes SOD, CAT and GPx), biotransformation (GSTs) and lipid peroxidation (TBARS), and histological assessment (sex determination and assessment of gonadal developmental stages).

Section snippets

Chemicals

Simvastatin (SIM) ([(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl] 2,2-dimethylbutanoate; CAS: 79902−63-9; purity ≥ 97 %) was acquired from Sigma-Aldrich (Schnellforf, Germany). All reagents were purchased from Sigma-Aldrich, except for Bradford reagent, which was purchased from Bio-Rad UK. The chemicals used for histological analyses were ethanol absolute (CAS: 64−17-5), acquired from AGA – Álcool e Géneros Alimentares S.A.;

Erratic swimming

According to the results obtained in terms of erratic swimming by fish exposed to SIM, there were significant differences between exposed groups and the control animals, for both (light and dark) periods. During the first light cycle (0–300 s), there was a significant decrease in erratic swimming, except for the organisms exposed to the second highest concentration (739.6 ng L−1), compared with control treatment (One-Way ANOVA on Ranks followed by a Dunn’s test: H5 = 91.651, p < 0.001). During

Behavioural assessment

According to the results obtained for erratic swimming, observed in individuals exposed to SIM, the general patterns observed consisted in a decrease in the first light period, an increase in the first dark cycle, and a decrease in the second light and dark periods, compared with the control group. Regarding the results obtained in terms of purposeful swimming in individuals exposed to SIM, the overall patterns reported were an increase in the first light period, a decrease in the first dark

Conclusions

Ecotoxicological data for lipid-regulating drugs, such as SIM, are still lacking, and these drugs have only been confusingly characterized in terms of modes of action and consequences in aquatic organisms. This assay provided information about the ecotoxicity of SIM, in both embryonic and juvenile stages of D. rerio. Results from this study demonstrate that zebrafish early-life stages and juvenile individuals can serve as model organisms in ecotoxicological assays (Scholz et al., 2008; Lammer

CRediT authorship contribution statement

D. Rebelo: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft. A.T. Correia: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - review & editing. B. Nunes: Conceptualization, Methodology, Validation, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Bruno Nunes is hired by “ECO-R-pharmplast - Ecotoxicity of realistic combinations of pharmaceutical drugs and microplastics in marine ecosystems”, Fundação para a Ciência e a Tecnologia, FCT (reference POCI-01-0145-FEDER-029203). This research was financially supported by CESAM (UIDB/50017/2020+UIDP/50017/2020), by CIIMAR (UIDB/04423/2020+UIDP/04423/2020), by FCT/MCTES through national funds (PIDDAC), and by the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.

References (117)

  • P. Chavan-Gautam et al.

    Distribution of fatty acids and lipids during pregnancy

    Adv. Clin. Chem.

    (2018)
  • J. Chen et al.

    TBBPA exposure during a sensitive developmental window produces neurobehavioral changes in larval zebrafish

    Environ. Pollut.

    (2016)
  • C.M.H. Combelles et al.

    Could oxidative stress influence the in-vitro maturation of oocytes?

    Reprod. Biomed. Online

    (2009)
  • U. Dahl et al.

    Application of growth-related sublethal endpoints in ecotoxicological assessments using a harpacticoid copepod

    Aquat. Toxicol.

    (2006)
  • D. Delwing-de Lima et al.

    Guanidino compounds inhibit acetylcholinesterase and butyrylcholinesterase activities: effect neuroprotector of vitamins E plus C

    Int. J. Dev. Neurosci.

    (2010)
  • V.B. Djordjević

    Free radicals in cell biology

    Int. Rev. Cytol.

    (2004)
  • K. Dooley et al.

    Zebrafish: a model system for the study of human disease

    Curr. Opin. Genet. Dev.

    (2000)
  • J.P. Dzoyem et al.

    Biochemical parameters in toxicological studies in Africa: significance, principle of methods, data interpretation, and use in plant screenings

    Toxicol. Surv. Afr. Med. Plants

    (2014)
  • A.J. Ebele et al.

    Pharmaceuticals and personal care products (PPCPs) in the freshwater aquatic environment

    Emerg. Contam.

    (2017)
  • G. Federici et al.

    Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other physiological effects

    Aquat. Toxicol.

    (2007)
  • L. Flohé et al.

    Superoxide dismutase assays

    Meth. Enzymol.

    (1984)
  • E. Gracia-Lor et al.

    Occurrence and removal of pharmaceuticals in wastewater treatment plants at the Spanish Mediterranean area of Valencia

    Chemosphere

    (2012)
  • C. Green et al.

    Environmental concentrations of anti-androgenic pharmaceuticals do not impact sexual disruption in fish alone or in combination with steroid oestrogens

    Aquat. Toxicol.

    (2015)
  • W.H. Habig et al.

    Glutathione S-transferases. The first enzymatic step in mercapturic acid formation

    J. Biol. Chem.

    (1974)
  • T.F. Holth et al.

    Differential gene expression and biomarkers in zebrafish (Danio rerio) following exposure to produced water components

    Aquat. Toxicol.

    (2008)
  • A. Jelic et al.

    Occurrence, partition and removal of pharmaceuticals in sewage water and sludge during wastewater treatment

    Water Res.

    (2011)
  • B. Kasprzyk-Hordern et al.

    The removal of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs during wastewater treatment and its impact on the quality of receiving waters

    Water Res.

    (2009)
  • E. Lammer et al.

    Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test?

    Comp. Biochem. Physiol. Part C Toxicol. Pharmacol.

    (2009)
  • J. Lykkesfeldt

    Malondialdehyde as biomarker of oxidative damage to lipids caused by smoking

    Clin. Chim. Acta

    (2007)
  • J. Martín et al.

    Comparison of different extraction methods for the determination of statin drugs in wastewater and river water by HPLC/Q-TOF-MS

    Talanta

    (2011)
  • K.A. Modesto et al.

    Roundup® causes oxidative stress in liver and inhibits acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus

    Chemosphere

    (2010)
  • L.R. Murphy et al.

    Physiological effects of short-term salinity changes on Ruppia maritima

    Aquat. Bot.

    (2003)
  • T. Neuparth et al.

    Hypocholesterolaemic pharmaceutical simvastatin disrupts reproduction and population growth of the amphipod Gammarus locusta at the ng/L range

    Aquat. Toxicol.

    (2014)
  • L.L.D. Oliveira et al.

    Evaluation of ecotoxicological effects of drugs on Daphnia magna using different enzymatic biomarkers

    Ecotoxicol. Environ. Saf.

    (2015)
  • P. Paíga et al.

    Assessment of 83 pharmaceuticals in WWTP influent and effluent samples by UHPLC-MS/MS: hourly variation

    Sci. Total Environ.

    (2019)
  • M. Papageorgiou et al.

    Seasonal occurrence, removal, mass loading and environmental risk assessment of 55 pharmaceuticals and personal care products in a municipal wastewater treatment plant in Central Greece

    Sci. Total Environ.

    (2016)
  • A. Piechota-Polanczyk et al.

    Simvastatin decreases free radicals formation in the human abdominal aortic aneurysm wall via NF-κB

    Eur. J. Vasc. Endovasc. Surg.

    (2012)
  • P.D. Ray et al.

    Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling

    Cell. Signal.

    (2012)
  • A. Ayala et al.

    Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal

    Oxid. Med. Cell. Longev.

    (2014)
  • S. Banerjee et al.

    Drug metabolism and oxidative stress: cellular mechanism and new therapeutic insights

    Biochem. Anal. Biochem. s3

    (2016)
  • R. Basnet et al.

    Zebrafish larvae as a behavioural model in neuropharmacology

    Biomedicines

    (2019)
  • R. Bogers

    Markers of Endocrine Disruption in Fish

    (2008)
  • J. Bouayed

    Relationship between oxidative stress and anxiety: emerging role of antioxidants within therapeutic or preventive approaches

    In Anxiety Disorders

    (2011)
  • A.B.A. Boxall

    The environmental side effects of medication

    EMBO Rep.

    (2004)
  • M. Broncel et al.

    The effect of statins on lipids peroxidation and activities of antioxidants enzymes in patients with dyslipidemia

    Prz. Lek.

    (2006)
  • A.A. Bustan et al.

    The effect of two types of statins (Rosuvastatin and atorvastatin) on the fertility of male and female mice

    Br. J. Med. Med. Res.

    (2017)
  • E.J. Calabrese et al.

    U-shaped dose-responses in biology, toxicology, and public health

    Annu. Rev. Public Health

    (2001)
  • L.M. Campos et al.

    Alterations in zebrafish development induced by simvastatin: comprehensive morphological and physiological study, focusing on muscle

    Exp. Biol. Med.

    (2016)
  • V. Cunha et al.

    Simvastatin effects on detoxification mechanisms in Danio rerio embryos

    Environ. Sci. Pollut. Res.

    (2016)
  • D. Dagneaux et al.

    Detecting general patterns in fish movement from the analysis of fish tagging data

    18 th World IMACS / MODSIM Congress

    (2009)
  • Cited by (0)

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